Application of NLRP3 inhibitor in preparation of medicine for treating biliary atresia
By using NLRP3 inhibitors to inhibit the inflammatory pathway in cholangioinflammatory injury, the problem of cholangioinflammatory injury in bile tract atresia was solved, and liver function and bile duct injury were significantly improved, and survival rate was improved.
Patent Information
- Application Number
- CN202510260598.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art is difficult to effectively solve the pathological mechanism of cholangioinflammatory injury in biliary atresia, and the treatment methods are limited.
NLRP3 inhibitors or their derivatives are used to selectively inhibit the inflammasome NLRP3 activated by activation of abnormal pan-apoptotic pathways, thereby inhibiting the proinflammatory factor product IL-1β, and alleviating the formation of bile duct atresia and inflammation.
Effectively reduces the jaundice rate, improves weight and survival rate, significantly improves liver function and bile duct damage, and provides a new potential strategy for the treatment of biliary atresia.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the use of NLRP3 inhibitors in the preparation of drugs for treating biliary atresia. Background Art
[0002] Biliary atresia (BA) is caused by inflammatory and fibrotic obstruction of extrahepatic bile ducts and is the leading indication for pediatric liver transplantation worldwide. At the time of diagnosis, the extrahepatic bile ducts are completely obstructed. At the tissue level, there is segmental or global loss of the inner layer of extrahepatic bile duct epithelium, accompanied by extensive fibrosis and occasional focal inflammation. In contrast, intrahepatic bile ducts are typically hyperplastic, embedded in portal tracts containing variable inflammation and fibrosis, and surrounded by lobules characterized by cholestasis and varying degrees of giant multinucleated hepatocytes. The clinical features of the disease are simple and reproducible: pathologic jaundice with direct or conjugated hyperbilirubinemia, acholic stools, varying degrees of hepatosplenomegaly, and the onset of symptoms is limited to the first few months after birth. When the disease is untreated, progression to end-stage cirrhosis is consistent. The initial goal of clinical treatment is timely diagnosis so that surgical intervention can remove the remaining remnants of the atretic bile ducts and establish a Roux-en-Y intestinal tube for bile drainage, also known as Kasai portoenterostomy / hepatoportoenterostomy. Unfortunately, unacceptably, 40-50% of patients still do not improve bile drainage after portoenterostomy; even if successful improvement is achieved, most patients require liver transplantation to extend their survival.
[0003] Clinical findings have shown that the progression of biliary atresia is closely related to progressive inflammatory damage of bile ducts. However, the specific triggering factors are currently unclear and the treatment options are limited. Therefore, it is urgent to clarify the pathological mechanism of the occurrence of inflammatory damage in biliary atresia bile ducts, and there is an urgent need for new strategies for specific prevention and treatment of bile duct inflammatory damage. Summary of the Invention
[0004] The present invention aims to at least solve one of the above technical problems existing in the prior art. To this end, the object of the present invention is to provide the use of NLRP3 inhibitors in the preparation of drugs for treating biliary atresia.
[0005] To achieve the above object, the technical solution adopted by the present invention is:
[0006] The present invention provides the use of NLRP3 inhibitors or their derivatives in the preparation of drugs for treating biliary diseases.
[0007] In biliary atresia, myeloid-derived suppressor cells (M-MDSCs) activate CD177+ cells to secrete neutrophil extracellular traps (NETs) through TNF-α and IL-1β, thereby causing bile duct injury. Further analysis showed that the pan-apoptosis pathway in M-MDSCs was activated, leading to the secretion of a large amount of NLRP3 and IL-1β by M-MDSCs. IL-1β activates CD177+ cells to secrete a large amount of NETs, resulting in bile duct injury. At the same time, IL-1β can also attract more immune cells to the infection site, causing persistent inflammation around the bile ducts. Molecules in the PANoptosis pathway, including NLRP3, IL-1β, etc., may be targets for preventing cytokine storms, which opens up a new way for targeted therapeutic interventions. In the present invention, the NLRP3 inhibitor can selectively inhibit the inflammasome NLRP3 produced by the activation of the abnormal pan-apoptosis pathway, thereby inhibiting the production of the pro-inflammatory factor IL-1β, effectively alleviating the formation of biliary atresia in BA mice, reducing the jaundice rate, and increasing body weight and survival rate.
[0008] In some embodiments of the present invention, the NLRP3 inhibitor includes at least one of Dapansutrile (OLT1177), BMS-986299, SB-414, MCC-950, IFM-514, JT-194, PELA-167, NBC-6, or a pharmaceutically acceptable salt thereof. In the present invention, Dapansutrile can effectively improve the liver function indexes of BA mice, significantly reduce the liver inflammation level, and significantly improve bile duct injury.
[0009] In some embodiments of the present invention, the biliary diseases include biliary atresia, cholangitis, cholecystitis, cholelithiasis, biliary parasitosis, and jaundice.
[0010] In some embodiments of the present invention, the biliary atresia also presents as food allergy caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver diseases caused by biliary atresia, and intestinal diseases caused by biliary atresia.
[0011] In some embodiments of the present invention, the liver diseases caused by biliary atresia include liver function injury caused by biliary atresia and / or liver inflammatory diseases caused by biliary atresia.
[0012] In some embodiments of the present invention, the intestinal diseases caused by biliary atresia include intestinal inflammatory diseases caused by biliary atresia.
[0013] In some embodiments of the present invention, the jaundice includes pathological jaundice or neonatal jaundice.
[0014] In some embodiments of the present invention, the pathological jaundice includes pathological jaundice caused by viruses or pathological jaundice in infancy.
[0015] In some embodiments of the present invention, the pathological jaundice further includes pathological jaundice in infancy caused by viruses; or the pathological jaundice caused by viruses includes cytomegalovirus jaundice.
[0016] In some embodiments of the present invention, the cholangitis includes bacterial cholangitis, viral cholangitis, cholangitis caused by biliary atresia, and cholangitis complicated after biliary atresia surgery.
[0017] In some embodiments of the present invention, the bacterial cholangitis further includes bacterial cholangitis caused by biliary atresia and bacterial cholangitis complicated after biliary atresia surgery.
[0018] In some embodiments of the present invention, the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults; preferably, the children include neonates within 28 days after birth, infants within 1 year old, toddlers aged 1 - 6 years old, and children aged 6 - 18 years old; preferably, the adults include female adults during pregnancy, perinatal female adults, and lactating female adults.
[0019] In some embodiments of the present invention, the NLRP3 inhibitor derivatives include NLRP3 inhibitors such as Dapansutrile derivatives including pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers, prodrugs, and Dapansutrile functional equivalents.
[0020] In some embodiments of the present invention, the drug further includes other active ingredients, and the other active ingredients include at least one preparation or compound known in the art for the treatment of biliary diseases, such as at least one of antibiotics, folic acid, ursodeoxycholic acid, phenobarbital, cholestyramine, and PDE inhibitors (such as dipyridamole).
[0021] In some embodiments of the present invention, the dosage form of the drug includes capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, granules, honeyed refined creams, sustained - release and controlled - release preparations, oral liquid preparations, injections, chewable tablets, oral tablets, transdermal patches, and effervescent tablets; or, the dosage form of the drug includes dosage forms for administration via the gastrointestinal tract or dosage forms for non - gastrointestinal administration.
[0022] In some embodiments of the present invention, the dosage forms for administration via the gastrointestinal tract include powders, tablets, granules, capsules, sustained - release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, etc.
[0023] In some embodiments of the present invention, the parenteral dosage forms include injection dosage forms (such as injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (such as sprays, aerosols, powder aerosols, etc.); skin dosage forms (such as topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (such as eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets, adhesive tablets, film dressings, etc.); and cavity dosage forms (such as suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).
[0024] In some embodiments of the present invention, the unit dose of the NLRP3 inhibitor or its derivative in the drug is 0.1 mg to 1 g; preferably, the unit dose of the NLRP3 inhibitor or its derivative in the drug is 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, 5.0 mg, 6.0 mg, 6.5 mg, 7.0 mg, 7.5 mg, 8.0 mg, 8.5 mg, 9.0 mg or 10.0 mg; preferably, the unit dose of the NLRP3 inhibitor or its derivative in the drug is 2.0 mg, 4.0 mg, 6.0 mg, 8.0 mg or 10.0 mg.
[0025] The beneficial effects of the present invention are as follows:
[0026] The NLRP3 inhibitor can selectively inhibit the inflammasome NLRP3 generated by the activation of the abnormal pan-apoptosis pathway, thereby inhibiting the production of the pro-inflammatory factor IL-1β, effectively alleviating the formation of biliary atresia in BA mice, reducing the jaundice rate, and increasing the body weight and survival rate.
[0027] Dapansutrile has a certain therapeutic effect on biliary atresia, can significantly prolong its survival time, improve its liver function, and reduce the number of inflammatory cell infiltrations around the intrahepatic bile ducts, providing a new potential target and treatment strategy for the treatment of biliary atresia. Description of the Drawings
[0028] Figure 1 Shows the survival status of mice in each group in the embodiments of the present invention; among them, A is the appearance diagram of mice in each group on the 12th day; B is the fluorescence angiogram of the extrahepatic bile duct of mice in each group on the 12th day.
[0029] Figure 2 Shows the body weight curves of mice in each group in the embodiments of the present invention.
[0030] Figure 3 Shows the jaundice rate diagram of mice in each group in the embodiments of the present invention.
[0031] Figure 4 Survival curves of mice in each group in the embodiments of the present invention.
[0032] Figure 5 Liver function indexes of mice in each group on the 12th day, where A is alanine aminotransferase (ALT); B is aspartate aminotransferase (AST); C is alkaline phosphatase (ALP); D is γ-glutamyl transpeptidase (γ-GT); E is total bilirubin (TBIL); F is direct bilirubin (DBIL); G is total bile acid (TBA).
[0033] Figure 6 Results of the effects of Dapansutrile on portal area inflammation infiltration and intrahepatic bile ducts in BA mice, where A is the H&E staining result diagram of the liver of mice in each group on the 12th day; B is the inflammation cell infiltration area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of mice in each group on the 12th day; C is the bile duct epithelial cell area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of mice in each group on the 12th day. Detailed implementation manners
[0034] The content of the present invention will be further described in detail through specific embodiments below. The raw materials, reagents or devices used in the embodiments and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.
[0035] Embodiment
[0036] This embodiment tests the therapeutic effect of Dapansutrile on BA mice. The specific process is as follows:
[0037] (I) Experimental materials:
[0038] Experimental animals: BALB / c WT neonatal mice within 24 hours after birth.
[0039] Main reagents and antibodies:
[0040] (1) Rhesus rotavirus (RRV) MMU18006, with a titer of 1.5×10 6 PFU / mL and a dose of 20 μL.
[0041] (2) Dapansutrile (MCE, HY-17629)
[0042] (II) Experimental methods:
[0043] Experimental steps:
[0044] Animal grouping: BALB / c WT neonatal mice within 24 hours after birth were randomly divided into 4 groups: normal control group (Saline group, physiological saline group), disease model group (RRV group, experimental group), and intervention group (RRV+Dapansutrile group, drug treatment group).
[0045] Injection method: Treatment of the RRV group: Within 24 hours after birth of BALB / c neonatal mice, 20 μL of RRV (titer: 1.5×10 6 PFU / mL) was intraperitoneally injected using a disposable sterile insulin syringe to induce BA formation; Treatment of the Saline group: Within 24 hours after birth of the mice, 20 μL of physiological saline was intraperitoneally injected using a disposable sterile insulin syringe; Treatment of the RRV+Dapansutrile group: After injecting RRV into the mice within 24 hours after birth, starting from the second day, Dapansutrile (dose: 100 mg / kg) was intraperitoneally injected using a disposable sterile insulin syringe once a day until the 12th day.
[0046] 1. Observe and record the survival status, survival body weight, and skin jaundice of the mice in each group every day, and collect blood and liver tissue samples on the 12th day; continue to observe the parallel groups.
[0047] 2. Fluorescent cholangiography of extrahepatic bile ducts: On the 12th day after birth of the mice in each group, 2% pentobarbital sodium (40 mg / kg) and buprenorphine (0.05 mg / kg) were used for anesthesia and analgesia. On a clean microscopic operating table, dissection was performed using sterile forceps and scissors to fully expose the liver, gallbladder, and extrahepatic bile ducts of the mice. An insulin syringe filled with the fluorescent contrast agent solution was inserted into the gallbladder cavity, and the contrast agent was slowly injected. Whether the contrast agent passed through the extrahepatic bile ducts to the jejunum was observed under the microscope and photographed.
[0048] 3. Blood collection and biochemical detection: Cardiac blood collection was performed on the 12th day after birth of the neonatal mice. When collecting blood, the mice were anesthetized by inhaling isoflurane. On a clean microscopic operating table, the abdominal skin was picked up with forceps, and the abdomen and chest were cut open with scissors to fully expose the diaphragm of the mice. A small incision was made on the left side of the diaphragm with scissors to expose the heart. An insulin syringe was inserted into the apex of the mouse's heart and blood was slowly drawn following the heart rhythm (after inserting the needle into the apex, there was an obvious breakthrough feeling, indicating entry into the left ventricle) until no more blood could be drawn. The drawn blood was transferred into an anticoagulation tube, labeled, and centrifuged at 3000 rpm for 10 minutes at room temperature to separate the serum. The separated serum was transferred to a new EP tube. Serum with a volume less than 120 μL was diluted to 120 μL with PBS, and the dilution factor was recorded. Subsequently, the serum was stored in a -30°C refrigerator for future detection. The serum to be tested was taken to the hospital laboratory for liver function testing using a biochemical detection instrument.
[0049] 4. H&E staining: Fix the fresh mouse liver tissues of each group on the 12th day in 10% formalin overnight, then embed them in paraffin and section. The sections are successively dewaxed, hydrated, stained with hematoxylin, differentiated with 1% hydrochloric acid alcohol, and stained with eosin. Finally, observe the pathological changes of the liver tissues under a microscope.
[0050] 5. CK19 immunohistochemical staining: Dewax and hydrate the liver tissue sections, immerse the sections in Tris-EDTA buffer (pH 9.0), heat them in a microwave oven at 95 °C for 10 minutes for antigen retrieval. Expose the sections to 3% hydrogen peroxide solution for 10 minutes to remove endogenous peroxidase. Treat the sections with 5% goat serum to block non-specific binding. Add rabbit-mouse CK19 primary antibody (diluted 1:200) to the sections and incubate overnight at 4 °C. Incubate the sections with the appropriate secondary antibody at room temperature for 30 minutes. Use 3,3'-diaminobenzidine (DAB) as the chromogen to visualize the immunohistochemical staining. Observe the sections under a microscope, acquire images, and analyze as needed.
[0051] Observation indicators and detection methods:
[0052] 1. Observation of general conditions of mice: Observe and record the survival status, body weight, skin jaundice, and the color of urine and feces of each group of mice every day.
[0053] 2. Mouse dissection and sample collection: On the 12th day, euthanize and dissect the mice, observe the appearance of the liver and bile ducts, and perform fluorescence angiography of the extrahepatic bile ducts using a fluorescent contrast agent.
[0054] 3. Detection of liver function indicators: Use a biochemical analyzer to detect liver function indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and total bile acid (TBA) in the serum of mice.
[0055] 4. Histopathological examination of liver tissues: Fix, embed, and section the liver tissues, and perform H&E staining and CK19 immunohistochemical staining. H&E staining can observe the infiltration of inflammatory cells around the intrahepatic bile ducts, and CK19 immunohistochemical staining can observe the damage of the intrahepatic bile ducts.
[0056] Figure 1 Figure A shows the appearance of each group of mice on the 12th day; Figure 1 Figure B is the fluorescence angiogram of the extrahepatic bile ducts of each group of mice on the 12th day; Figure 2 is the body weight curve of each group of mice (days 0 - 20); Figure 3 is the jaundice rate graph of each group of mice (days 0 - 20); Figure 4Survival curves of mice in each group (days 0 - 20).
[0057] It can be seen that the jaundice rate of mice in the RRV group was greater than 90%, indicating that the above-mentioned RRV treatment could successfully establish a model; in the RRV group of mice treated with Dapansutrile (OLT1177), the skin jaundice of BA symptoms was significantly improved ( Figure 1 in A), the jaundice rate decreased ( Figure 3 ), the extrahepatic bile duct was completely unobstructed ( Figure 1 in B), the decrease in the average survival body weight slowed down significantly ( Figure 2 ), and the survival time was significantly prolonged ( Figure 4 ).
[0058] Figure 5 Liver function indexes of mice in each group on the 12th day. Among them, Figure 5 A in it is alanine aminotransferase (ALT); Figure 5 B in it is aspartate aminotransferase (AST); Figure 5 C in it is alkaline phosphatase (ALP); Figure 5 D in it is γ-glutamyl transpeptidase (γ-GT); Figure 5 E in it is total bilirubin (TBIL); Figure 5 F in it is direct bilirubin (DBIL); Figure 5 G in it is total bile acid (TBA).
[0059] It can be seen that compared with the RRV group, the liver function of BA mice was significantly improved after treatment with Dapansutrile (OLT1177).
[0060] Figure 6 Results of the effects of Dapansutrile (OLT1177) on inflammatory cell infiltration in the portal area and intrahepatic bile ducts of BA mice. Among them, Figure 6 A in it is the H&E staining result diagram of the liver of mice in each group on the 12th day; Figure 6 B in it is the inflammatory cell infiltration area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of mice in each group on the 12th day; C is the bile duct epithelial cell area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of mice in each group on the 12th day.
[0061] It can be seen that on the 12th day, the inflammatory cell infiltration around the intrahepatic bile ducts of mice in the RRV group increased significantly, and the intrahepatic bile ducts were blocked; compared with the RRV group of mice, the inflammatory cell infiltration around the intrahepatic bile ducts of RRV mice treated with Dapansutrile (OLT1177) decreased significantly, there was a normal intrahepatic bile duct structure, and the degree of bile duct injury was significantly reduced.
[0062] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.
Claims
1. Use of NLRP3 inhibitors or their derivatives in the preparation of drugs for the treatment of biliary diseases.
2. The use according to claim 1, characterized in that: The NLRP3 inhibitor includes at least one of Dapansutrile, BMS-986299, SB-414, MCC-950, IFM-514, JT-194, PELA-167, NBC-6 or a pharmaceutically acceptable salt thereof.
3. The use according to claim 1, characterized in that: The biliary diseases include biliary atresia, cholangitis, cholecystitis, cholelithiasis, biliary parasitic diseases, and jaundice.
4. The use according to claim 3, characterized in that: The biliary atresia may also manifest as food allergies caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver diseases caused by biliary atresia, and intestinal diseases caused by biliary atresia.
5. The use according to claim 3, characterized in that: The jaundice includes pathological jaundice or infantile jaundice.
6. The use according to claim 3, characterized in that: The cholangitis includes bacterial cholangitis, viral cholangitis, cholangitis caused by biliary atresia, and cholangitis complicated by biliary atresia surgery.
7. The use according to claim 1, characterized in that: The dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults; preferably, the children include newborns within 28 days of birth, infants under 1 year old, toddlers aged 1 to 6 years old, and children aged 6 to 18 years old; preferably, the adults include female adults in pregnancy, female adults in the perinatal period, and female adults in lactation.
8. The use according to claim 1, characterized in that: The medicine further comprises other active ingredients, and the other active ingredients include at least one of antibiotics, folic acid, ursodeoxycholic acid, phenobarbital, cholestyramine, and a PDE inhibitor.
9. The use according to claim 1, characterized in that: The dosage forms of the drug include capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, injections, chewable tablets, buccal tablets, transdermal patches and effervescent tablets; or, the dosage forms of the drug include dosage forms for gastrointestinal administration or non-gastrointestinal administration.
10. The use according to claim 1, characterized in that: The unit dosage of the NLRP3 inhibitor or its derivative in the medicine is 0.1 mg to 1 g.